Field of the Invention
In the mechanisms of differentiation and proliferation of cells, membrane proteins expressed on the surface of said cells have a very important biological significance. Cytokine, peptide hormone or neurotransmitter receptors, cell adhesion molecules and the like are involved in signal transduction from cell to cell. The present invention relates to a method of cloning proteins coding for such membrane proteins without requiring any antibody or ligand at all. More particularly, it relates to a method of cloning proteins having transmembrane domains.
The methods most widely used at present for cloning membrane molecules include several improvements or modifications of the expression (or phenotype) cloning method designed in 1987 by Seed and Aruffoi (Proc. Acad. Sci. USA, 84:3365-3369, 1987). They comprise utilizing, for example, transient expression by Cos7 cells and an expression vector containing the replication origin of SV40, thus introducing a cDNA library constructed using an expression vector for use in animal cells into Cos7 cells, and thereby causing expression of a cDNA library-derived membrane protein on the cell surface. Cells in which the desired target membrane molecule is expressed are concentrated by panning or by means of a FACS, using an antibody to or a ligand of said membrane molecule, and the plasmid DNA is recovered and used again to transfect Cos7 cells, and this concentration procedure is repeated (panning method or sorting method, respectively). Another method comprises dividing an expression vector cDNA library into a certain number of small groups, introducing each group into Cos7 cells, detecting membrane molecule expression using an isotope-labeled antibody or ligand, dividing the positive group or groups further into small groups each comprising a small number of plasmids, and repeating the transfection to attain cloning (sib selection method).
However, the methods mentioned above each is effective only when the antibody to or the ligand of the target membrane molecule expressed on the cell surface is clearly known. On the contrary, the present method is an epoch-making one which enables cloning of a membrane molecule on cells without requiring any antibody or ligand at all. The fact that no antibody or ligand is required is very advantageous in that intracellular signal transduction substances can be cloned while avoiding troublesome establishment of assay systems in which a biological activity is used as an indicator. Further, in the case of cytokine receptors, the level of expression on the cell surface is very low (several hundreds to several thousands per cell) and, therefore, it is difficult to prepare monoclonal antibodies which are to be obtained through immunization with a solublized cell fraction. In addition, it is practically impossible to make approaches by the conventional method based on amino acid sequence determination following protein purification. Unlike the conventional methods, the present method is a novel one which enables even cloning of quite novel membrane molecules the ligand of which has never been identified.
The present invention consists in a method of cloning a DNA coding for a membrane protein which comprises the steps of:
A) selecting a protein having a signal sequence, and preparing a DNA (reporter gene) coding for the signal sequence and N terminal sequence of said protein;
B) constructing a DNA library containing a DNA coding for a target membrane protein;
C) constructing an expression vector by joining the DNA library obtained in B) above to the downstream of the reporter gene obtained in A) above;
D) transforming an appropriate host with the expression vector obtained in C) above, and selecting cells in which the reporter gene is expressed on the cell surface; and
E) recovering the vector from the positive cells obtained to give a clone containing a part or the whole of the DNA coding for the target membrane protein; and
F) when the DNA obtained in E) above codes only for a part of the target protein, obtaining a complete DNA clone based on the sequence thereof.
The present invention also relates to the cDNA obtained by using these methods, a vector containing said DNA, a transformant resulting from transformation with said vector, and the product of transcription of said DNA as produced by said transformant.
The present inventors revealed that even a membrane molecule which is quite new and an antibody to which can hardly be prepared or any ligand of which has not yet been identified can be cloned by using the method of the present invention.
In the following, the cloning method of the present invention is fully described step by step.
A) First of all, a protein having a signal sequence is selected, and a DNA (reporter gene) coding for the signal sequence and N-terminal sequence of said protein is prepared. The signal sequence-containing protein is such a protein as to permeate through the membrane and includes, among others, membrane proteins and secretory proteins. As the reporter gene, there may be mentioned, for example, a DNA obtained by selecting a known membrane protein and depriving a DNA coding for, said membrane protein of the transmembrane domain-encoding region and the downstream thereof. Said known membrane protein is not limited to any particular protein provided that the nucleotide sequence coding therefor is already known (for example, the cDNA sequence for said protein is known, or the chromosome DNA sequence is known) and thus the transmembrane domain(s) thereof can be estimated and that the expression thereof on the membrane surface can be confirmed. It is, however, preferred that it be a homologous protein (for example, if the novel membrane protein to be cloned (target membrane protein) is a mammal-derived one, the known membrane protein should be also a mammal-derived one). As specific examples of such known membrane protein, there may be mentioned known mammalian cell-surface antigens, such as murine L3T4, human CD4 and human HLA antigens, and various receptor proteins occurring on the cell surface, such as receptor type tyrosine kinase, receptor type serine/threonine kinase and cytokine receptors. A DNA corresponding to one derived from the DNA coding for such a known membrane protein by deletion of the transmembrane domain(s) and the downstream thereof is prepared by utilizing an ordinary genetic engineering technique (e.g. DNA synthesis, restriction enzyme cleavage of cDNA followed by purification, etc.).
B) A DNA library containing a DNA coding for the target membrane protein is then constructed. The DNA library containing a DNA coding for the target membrane protein may be a cDNA library or a chromosomal DNA library. The DNA library can be constructed by an ordinary method used in genetic engineering. For example, if a novel membrane protein expressed in hepatocytes is the target membrane protein, mRNA is collected from hepatocytes. If a novel membrane protein expressed in brain cells is the target membrane protein, mRNA is obtained from brain cells. Then, cDNA is produced using a reverse transcriptase and further converted to the corresponding double-stranded molecule using a DNA polymerase. A cDNA library is constructed by inserting said double-stranded molecule into an appropriate vector (e.g. plasmid or phage) A commercially available DNA library may also be used. When it is intended to obtain a cDNA for a membrane protein of a particular family gene origin, it is advantageous to use a cDNA library concentrated by degenerated PCR utilizing a consensus sequence such as a motif sequence, which occurs in the family genes.
C) And, an expression vector is constructed with the DNA of the DNA library obtained in B) above joined to the downstream of the reporter gene obtained in A) above. As specific examples of the vector to be used in constructing such expression vector, there may be mentioned pCD (Okayama et al., Mol. Cell. Biol., 3:280, 1983), pCD-SR (Takebeet, Mol. Cell. Biol., 8:466, 1988), pCDM 8 (Funakoshi), pBMG, pEF, pMAM (Toyobo) and the like. The expression vector is constructed by ligating the reporter gene and the DNA of the library containing a target membrane protein-encoding DNA, or a DNA derived therefrom by random hydrolysis, in that order, to the downstream of the promoter occurring in such a vector. When a CDNA sequence concentrated by degenerated PCR utilizing a consensus sequence is used, the cDNA can be joined in advance to the reporter gene without reading frame shifting.
D) An appropriate host is transformed with the expression vector obtained in C) above, and cells expressing the reporter gene on the membrane surface are selected. When the DNA coding for the transmembrane domain(s) in the DNA library is one encoding a fusion protein with a known membrane protein, said fusion protein is trapped on the cell membrane and the cells are rendered positive to an antibody against the epitope of the known membrane protein on the host cell membrane surface. In the case of a cDNA which does not code for any transmembrane domain, the fusion protein with a known membrane protein is not hold on the cell membrane, namely it is a secretory protein, hence it is not stained with an antibody against the known membrane protein. Therefore, by selecting cells binding to an antibody against the known membrane protein, it is possible to select those cells transformed with the expression vector containing a cDNA coding for the whole or part of the target membrane protein. In cases where the target membrane protein is of an animal cell origin, for instance, animal cells are preferred as the host cells. For example, such hosts as COS7, which can realize transient expression, are preferred. For selecting cells expressing the reporter gene on the membrane surface, the panning method, which utilizes an antibody against the reporter protein, or the sorting method using a FACS or the like is used, among others. These procedures for transformation, expression and selection may be repeated several times, as necessary, whereby the transformant (positive cells) expressing the target membrane protein can be concentrated.
E) The vector is recovered from the positive cells obtained to give a clone containing a part or the whole of the DNA coding for the target membrane protein. Where necessary, the base sequence of a fragment coding for the target membrane protein is determined and the sequence of the target membrane protein is analyzed or searched for using an analysis software of GENETYX (SOFTWARE-KAIHATSUGAISYA (software development company)), for instance, to determine whether the desired DNA has been cloned.
F) When the DNA obtained in E) above encodes only a part of the target membrane protein, a full DNA clone is obtained from a DNA library such as a cDNA library or a chromosomal DNA library on the basis of the sequence thereof.